Acetylation of histone H4 N-terminal tail plays a crucial role in gene activation in chromatin environments; and alterations in this epigenetic process can lead to various diseases. Our long-term objective is to understand how H4 acetylation regulates p53-dependent transcription to attenuate cancer development. To investigate these aspects, we have developed new protocols to interrogate the cellular functions of H4 acetylation as a signal that would facilitate the recruitment/retention of chromatin regulatory machinery at the site of gene transcription. Importantly, our development of recombinant chromatin transcription systems using wild type or mutated recombinant H4 proteins has allowed us to evaluate the contribution of each specific H4 acetylation event in p53-dependent transcription from chromatin. Our preliminary results from transcription analysis, coupled with chromatin acetylation assays, have indicated that acetylation of only a small subset of lysines in H4 tail is critical for transcriptional activation. To gain mechanistic insight into the function of H4 acetylation, we have established cell lines that stably express H4 tail domains for biochemical purification of acetylated H4 tail-associated factors. Importantly, our functional analysis revealed that the acetylated tail-associated factors could significantly enhance p53-dependent transcription. In light of specific interactions between acetylated H4 tails and regulatory factors, we also have generated cell-permeable H4 tail mimics bearing specific acetylation marks to screen for the most active H4 acetylation events in cellular transcription. Based on these results, our specific aims are (i) to investigate the specificity of H4 acetylation events at the level of p53-dependent transcription by checking inhibitory effects of mutations of H4 lysine substrates in vitro as well as by characterizing repressive actions of acetylated H4 tail mimics in vivo, (ii) to detail the molecular mechanism underlying H4 acetylation-mediated activation of p53-dependent transcription by isolating and characterizing acetylated H4 tail-associated factors that could counteract chromatin-induced repression of transcription, and (iii) to define the functional core of the H4 tail-associated factors by reconstituting the key regulatory components transcriptionally equivalent to the entire factors. Specific H4 tail-associated factors identified as playing crucial roles in p53-dependent transcription will be assigned as a target for the future design of therapeutic agents to block aberrant p53 regulation related to many human cancers. PUBLIC HEALTH RELEVANCE Our proposal describes multiple approaches to comprehensively understand how a specific chemical modification, acetylation of histone H4 protein within chromosomes, switches genes on in human cells. Since the regulation of this gene activation process is important in the control of cell growth and the establishment and maintenance of tissues and organs, the proposed studies should significantly contribute to our understanding of the molecular basis of many human diseases that are associated with uncontrolled gene expression.